Photosensitizer activity of phenothiazinium derivatives against melanoma cells and DFT insights into singlet oxygen generation.

The present study describes three novel phenothiazinium dyes with auxochrome variation designed to modulate their optical and biological properties. The synthesis of dyes 1-3 was based on the nucleophilic substitution of the phenothiazinium core group with aliphatic (1,2) or aromatic amines (3), and it was successfully conducted by classical, mechanochemical or sonochemical procedures. Their optical properties investigated by UV-vis absorption and fluorescence emission spectroscopy, revealed absorption maxima up to 645 nm and a strong negative solvatochromism in less polar solvents for the lipophilic derivative 3. The dyes 1 and 2 exhibited fluorescence emissions up to 690 nm, while the fluorescence of dye 3 was quenched in solution. Under irradiation at 660 nm using a light emitting diode, dyes 1-3 behaved as photosensitisers, demonstrating high capacity for singlet oxygen generation (quantum yields in the range 52 %-78 %). The theoretical DFT calculations and predicted Jablonski diagrams of photosensitisers 1 and 2 pointed out the triplet excited state involved in the process of energy transfer for singlet oxygen generation. The biological activity of photosensitizers 1-3 was explored on B16-F10 murine melanoma cells under light irradiation at 660 nm and indicated the highest inhibitory effect for dye 1 (IC = 29.2 μM), while dyes 2 (IC = 36.8 μM), and 3 (IC50 = 50.2 μM) were less cytotoxic. Their capacity to induce oxidative stress and unbalance the intracellular antioxidant defence system of the cells was evaluated by lactate dehydrogenase LDH, glutathione reductase and superoxide dismutase SOD assays. While the outcome of the LDH assay was similar for all three dyes, photosensitizers 2 and 3 exhibited a significant increase of SOD activity. Dye 1 displayed the strongest activating effect on glutathione reductase, while dye 3 presented the strongest effect on SOD.